Project Summary/Abstract From the unfertilized egg to specialized organs in the adult, the polarization of cells and intracellular structures is a fundamental aspect of animal development. In multicellular tissues, planar cell polarity (PCP) has been extensively studied in epithelia in Drosophila, however, comparatively little is known about the roles of cell polarity in developing mesenchymal tissues in vertebrates such as cartilage and bone. Cartilages composed of polarized stacks of chondrocytes give rise to a polarized progression of endochondral ossification. These stacks form through coordinated intercalation elongation, and differentiation within a skeletal condensation. Previously, we have shown that two PCP signaling pathways, Fat-Dachsous and Wnt-Fz, are required for the polarized stacking and differentiation of chondrocytes in the developing zebrafish jaw. A prominent feature of these polarized stacks that is disrupted in Fat3- or Wnt5-deficient embryos is the position of the microtubule- organizing center within each cell, which marks the location of the primary cilium. This suggests that the primary cilium may serve some specific function(s) during the formation of cartilages. Studies from both mouse and chick further implicate PCP and primary cilia in determining cartilage polarity in long bones. Moreover, defects in Wnt-Fz and Fat-Dachsous signaling cause human diseases. WNT5A or ROR2 mutations cause Robinow syndrome and FAT4 mutations cause Van Maldergem syndrome, both of which include skeletal defects. Similarly, diseases caused by mutations in cilia genes, collectively termed ?ciliopathies,? often present with skeletal defects, primarily affecting the face. These and other data from both mouse and chick suggest that defects in cell polarity underlie skeletal abnormalities including those affecting the craniofacial skeleton. Our preliminary data suggest a role for Hedgehog (Hh) signaling and primary cilia in chondrocyte polarity and for polarity in the formation of ?growth zones? (GZs), which are equivalent to long bone growth plates, and form during endochondral ossification at the sites of polarity reversals. We hypothesize that PCP and primary cilia function together to polarize chondrocytes in developing cartilages to establish sites of endochondral growth and ossification. In Aim 1 we will test novel requirements for the primary cilium and Hh signaling in cartilage polarity in zebrafish by 1) analyzing Fat-PCP and Wnt-PCP signaling responses to disrupting cilia and Hh signaling, 2) creating genetic mosaics with cell transplantation using cilia-deficient bbs/ofd1 mutants and Hh signaling-defective animals to explore cell non-autonomous effects of cilia and Hh on cartilage patterning, and 3) determining roles for Wnt- and Fat-PCP downstream of primary cilia signaling. In Aim 2 we will determine the roles of polarity in cartilage morphogenesis and GZ formation in zebrafish by 1) testing how cartilage polarity and primary cilia pattern GZs, 2) analyzing how polarity and primary cilia influence cartilage responses to Hh, and 3) studying potential cilia-independent roles for BBS9/OFD1 which may affect in cartilage polarity, including mitotic spindle pole orientation and cytokinesis in GZs.